Project Summary Mitochondria are required for a myriad of cellular functions. The mitochondrial network in cells is established and maintained by the coordinate activities of movement, fusion and division. Mitochondrial fusion in particular is beneficial as it increases cellular energy and protects against cell death. The molecular machines that mediate mitochondrial outer and inner membrane fusion are members of the dynamin superfamily. As such, they are large self-assembling GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. How these proteins couple self-assembly and the catalytic cycle to membrane tethering and lipid mixing is not known. The focus of this work is to examine the molecular mechanism of mitochondrial outer membrane fusion. Although Mitofusin1 (Mfn1) and Mitofusin2 (Mfn2) are both required for mitochondrial outer membrane fusion, they are functionally distinct. We discovered that mitochondrial fusion is most efficient when Mfn1 and Mfn2 are on opposite membranes. This suggests that they have unique molecular characteristics. To address this, we will compare and contrast Mfn1 and Mfn2 throughout our analyses and determine their unique biochemical properties. Our evaluation of the GTPase domain will reveal the mechanism of nucleotide binding, hydrolysis and release. We will reconstitute Mitofusin-dependent membrane tethering. This will allow us to determine the role of the catalytic cycle in membrane tethering and the role of lipid composition on Mitofusin tethering activity. Using disease associated missense mutations, we will identify unique features of the GTPase domain. We will identify the molecular determinants of Mitofusin complex assembly. We will combine cellular studies with powerful biochemical analyses, including reconstitution of lipid and content mixing with proteoliposomes. These studies will provide new insight into the unique mechanism of mitochondrial outer membrane fusion.